【作者】 罗会颖；

【导师】 范云六；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2008， 博士

【摘要】 极端微生物在生态系统中发挥着重要作用,为人类提供了丰富的微生物资源。随着越来越多的极端微生物被分离鉴定,极端酶被分离纯化和极端酶工程研究的进展,极端酶在生物催化和生物转化中的应用将会得到更进一步的拓展。本文对分离自江西“721”矿区铀矿废水的真菌MEY-1进行了初步鉴定。该菌具有极端嗜酸性,它的最适生长pH为2.5～3.0,在pH1.0下也能较好生长。经ITS序列比对并结合形态分析,MEY-1属于Bispora属。嗜酸真菌Bispora sp. MEY-1能分泌产生多种重要的工业用酶,其中包括木聚糖酶、β-葡聚糖酶、β-甘露聚糖酶、CMCase、淀粉酶、果胶酶、α-半乳糖苷酶、β-半乳糖苷酶以及β-葡萄糖苷酶等九种糖苷水解酶。是一株优良的工业用酶产生菌。初步建立了一种能诱导嗜酸真菌MEY-1同时产生上述九种酶的发酵方法。通过优化培养基中麸皮、玉米芯粉、魔芋粉及豆粕的组成比例,培养的pH和温度,建立了4种诱导不同酶产生的方法。方法1:麸皮、玉米芯粉、豆粕比例为2:5:2,pH 2.0,培养温度20℃,高产β-甘露聚糖酶、CMCase、淀粉酶、果胶酶。方法2:麸皮、玉米芯粉、豆粕比例为2:5:2,pH 2.0,培养温度30℃,高产β-葡聚糖酶。方法3:麸皮、玉米芯粉、豆粕比例为1:1:7,pH4.0,培养温度30℃高产α-半乳糖苷酶、β-半乳糖苷酶和β-葡萄糖苷酶。方法4:麸皮、玉米芯粉、豆粕比例为5:2:2,pH 4.0,培养温度30℃,高产木聚糖酶。本文通过糖苷水解酶基因序列分析,从嗜酸真菌Bispora sp. MEY-1分离克隆到7种9个糖苷水解酶的基因。分别是:木聚糖酶基因3个,β-甘露聚糖酶基因、α-淀粉酶基因、α-半乳糖苷酶基因、β-半乳糖苷酶基因、β-葡萄糖苷酶基因、α-L-鼠里糖苷酶基因各1个。通过BLAST比对和序列一致性分析,它们核苷酸和氨基酸序列的最高相似性分别为:木聚糖酶基因xyl11A, 85.3%和93.7%;木聚糖酶基因xyl11B, 61.4%和57.0%;木聚糖酶基因xyl10A, 49.8%和43.6%;β-甘露聚糖酶基因man5A, 45.4%和47.1%;α-淀粉酶基因amyA, 60.2%和62.0%;α-半乳糖苷酶基因agalA, 54.2%和49.7%;β-半乳糖苷酶基因bgalA,56.8%和55.5%;β-葡萄糖苷酶基因bglA,49.5%和34.8%;α-L-鼠里糖苷酶基因rhaA,55.0%和44.9%。并对其中的8个酶做了三维结构的模拟和催化位点的分析,结果表明,除xyl11A外,这些基因均具有较高的新颖性。将Bispora sp.MEY-1来源的β-甘露聚糖酶基因man5A,和3个木聚糖酶基因xyl11A, xyl11B, xyl10A在毕赤酵母中进行了表达。在摇床水平上β-甘露聚糖酶MAN5A的表达量为64 U/mL,木聚糖酶XYL11A,XYL11B和XYL10A的表达量分别为:83 U /mL,54 U /mL和101 U /mL。验证了基因的功能。对重组的β-甘露聚糖酶MAN5A和3个木聚糖酶进行了纯化和酶学性质分析。β-甘露聚糖酶MAN5A的最适pH为1.0～1.5,在pH1.0～5.5范围内酶活性均能维持在70%以上。这是目前报道的最适pH最低的β-甘露聚糖酶。三个木聚糖酶XYL11A,XYL11B和XYL10A的最适pH分别为2.4,2.6和3.0。均为极端嗜酸木聚糖酶。MAN5A的最适温度为65℃,木聚糖酶XYL11A,XYL11B和XYL10A的最适温度分别为60℃和65℃和80℃。他们都具有极好的抗胃蛋白酶和胰蛋白酶的能力,37℃条件下处理60min后的酶活力是处理前的85～150%。大部分金属离子不影响酶活力或对酶有激活作用。重组的β-甘露聚糖酶比活为3373U/mg,Km值为1.56mg/mL, Vmax为6587.6μmol/min·mg。三个重组木聚糖酶XYL11A,XYL11B和XYL10A的比活分别为8334U/mg,2049U/mg和5437U/mg。Km分别为19.907 mg/mL,29.96 mg/mL和1.005 mg/mL。Vmax为7308.5μmol/min·mg,1757.2μmol/min·mg和2709μmol/min·mg。结果表明该四个酶都是极端嗜酸酶,具有很高的比活性。综合酶学性质表明他们都具有很好的工业应用前景。更多还原

【Abstract】 Extremophiles play important roles in the ecosystem and provide rich microbial resources for human beings. Along with the isolation and identification of more and more extremophiles, various extremozymes have been isolated and purified, leading to the expansion of more applications of extremozymes in biocatalysis and biotransformation.In this study, an acidophilic fungus, MEY-1, was isolated from the acidic waste water of "721" Uranium Mine in Jiangxi, China. Isolate MEY-1 was able to grow at pH1.0～6.0, with the optimal growth at pH 2.5～3.0. Based on morphology and ITS sequence analysis, isolate MEY-1 was identified to belong to the genus Bispora. Being an excellent industrial enzyme producer, Bispora sp. MEY-1 can secrete nine important industrial enzymes, including xylanase,β-glucanase,β-mannanase, CMCase, amylase, pectinase,α-galactosidase,β-galactosidase andβ-glucosidaset.A fermentation system was initially established to induce MEY-1 to secrete the nine enzymes mentioned above. By optimizing the proportion of wheat bran, corncob powder, konjac flour and soybean meal in the medium, pH and temperature, four methods were used to induce different enzymes. Method 1 for high-yield ofβ-mannanase, CMCase, amylase and pectinase: the proportion of wheat bran, corncob powder and soybean meal was 2:5:2, pH 2.0 and 20℃. Method 2 for high-yield ofβ-glucanase: the proportion of wheat bran, corncob powder and soybean meal was the same as Method 1, pH 2.0 and 30℃. Method 3 for high-yield ofα-galactosidase,β-galactosidase andβ-glucosidase: the proportion of wheat bran, corncob powder and soybean meal was 1:1:7, pH4.0 and 30℃. Method 4 for high-yield of xylanase: the proportion of wheat bran, corncob powder and soybean meal was 5:2:2, pH 4.0 and 30℃.Based on sequence analysis, nine glucoside hydrolase genes belonging to seven kinds of enzymes were cloned from the acidophilic fungus Bispora sp. MEY-1. They are three xylanase genes, oneβ-Mannanase gene, oneα-amylase gene, oneα-galactosidase gene, oneβ- galactosidase gene, oneβ-glucosidase gene and oneα-L- rhamnosidase gene, respectively. Homology searches in GenBank using BLAST and multiple alignment using CLUSTALW were performed. The highest identity of nucleotide and amino acid sequences of these enzymes to known glucoside hydrolases are: xylanase gene xyl11A, 85.3% and 93.7%; xylanase gene xyl11B, 61.4% and 57.0%; xylanase gene xyl10A, 49.8% and 43.6%;β-mannanase gene man5A, 45.4% and 47.1%;α-amylase gene amyA, 60.2% and 62.0%;α-galactosidase gene agalA, 54.2% and 49.7%;β-galactosidase gene bgalA, 56.8% and 55.5%;β-glucosidase gene bglA, 49.5% and 34.8%;α-L- rhamnosidase gene rhaA, 55.0% and 44.9 %. Except for rhaA, all other enzymes were subject to three-dimensional structure and catalytic site analysis, indicating the novelty of eight enzymes besides xyl11A. Theβ-mannanase gene man5A, and three xylanase genes xyl11A, xyl11B and xyl10A were expressed in Pichia pastoris with the activity of 64, 83, 54 and 101 U/mL in flasks, verifying the function of these genes.The recombinantβ-mannanase MAN5A (MAN5A) and three xylanases (XYL11A, XYL11B and XYL10A) were purified and characterized. MAN5A had the optimum pH of 1.0~1.5, lower than all the knownβ-mannanases, and retained >70% activity over pH 1.0~5.5. The optimum pHs of XYL11A, XYL11B and XYL10A were 2.4, 2.6 and 3.0, respectively. The optimum temperatures of MAN5A, XYL11A, XYL11B and XYL10A were 65, 60, 65 and 80℃, respectively. All the recombinantβ-mannanase and three xylanases had strong resistance to pepsin and trypsin treatmen, retaining 85~150% activities at 37°C for 60 min. Metal ions and chemical reagents enhanced or had no effects on enzyme activities. The Km and Vmax values for MAN5A were 1.56 mg/mL and 6587.6μmol/min ? mg, respectively, with the specific activity of 3373 U/mg. The specific activity of XYL11A, XYL11B and XYL10A were 8334, 2049 and 5437 U/mg, respectivley. The Km and Vmax values for XYL11A, XYL11B and XYL10A were 19.907 and 7308.5, 29.96 and 1757.2, and 1.005 mg/mL and 2709μmol/min ? mg, respectively. All these four enzymes are acidicphilic with very high specific activities, suggesting their good prospects in industrial applications.更多还原